Typhoid is an endemic febrile disease caused by Salmonella typhi. Typhoid is a major concern of public health. The organism usually enters the body by consumption of contaminated food or water and penetrates the intestinal wall. After that it multiplies and enters blood stream within 24-72 hours resulting in enteric fever and bacteremia. After an incubation period of 10 to 14 days, early symptoms of typhoid, like headache, fever, loss of appetite, bradycardia, splenomegaly etc. appear. Typhoid in most cases is not fatal. Antibiotics, such as ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and ciprofloxacin, have been commonly used to treat typhoid in developed countries. Prompt treatment of the disease with antibiotics reduces the case-fatality rate to approximately 1%. When left untreated, typhoid fever persists for three weeks to a month. Death occurs in between 10% and 30% of untreated cases. Typhoid is diagnosed either by blood culture or by detection of its antigens or by the detection of its antibodies in the blood.
There are a number of limitations associated with diagnosis by widal test. The widal test is not specific as it cross reacts with other febrile organisms and many organisms of family Enterobacteriaceae. Further, as typhoid is an endemic disease, therefore, there always exists some background level of antibody in the endemic areas which gives misleading results in the widal test. Hence, it becomes necessary to determine the cut-off titer for each region to rule out the possibility of diagnosis as false positive. Further, the widal test gives positive results only after one or two weeks of the onset of fever. The widal test is that test is to be performed on paired serum samples taken at an interval of at least one week apart because single widal test is elusive and inconclusive.
Further limitation of the widal test is that the antibiotic administration in the early phase of infection, inhibits the development of the antibody and hence test may give false negative result. A further limitation of the widal test is that TAB vaccinated normal healthy persons give false positive reaction in widal test due to presence of circulating antibody against vaccine in human system. Another limitation of the widal test is that it gives indirect evidence of typhoid infection. Further limitation of the widal test is that the test has low sensitivity and low specificity.
Other technique known for diagnosis of typhoid is based upon isolation and identification causative agent. This procedure is termed as golden standard. In this technique Salmonella typhi is isolated from blood and identified by microscopic and biochemical test. However, this technique has many limitations. One limitation of the above technique is that it is time consuming as it requires long period of incubation from 3 days to 14 days and also requires elaborate laboratory facilities. Another limitation of the above technique is that for its performance large quantity of blood sample (10 ml/patient) is required. Yet another limitation of the above technique is that it needs large volume of culture medium i.e. 100 ml (10 times of blood sample). Still another limitation of the above technique is its low sensitivity (40 to 80%), as there are very few organisms in circulation, as low as 1/ml which leads to false negative results.
Further limitation of above method is that bacterial growth in culture is inhibited by serum bactericidal agents, present in blood which may lead to false negative results. Still further limitation of blood culture is that antibiotics treatment during early phase of infection may inhibit bacterial growth in culture which may give false negative results. WO2004047721 patent provides a process for the preparation of an agglutination reagent for rapid detection of typhoid. WO2007034508 provides an ultra-sensitive method for detection and/or quantification of an analyte in a sample.
Other known techniques such as Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay etc. are based on detection of circulating antigen in the body fluids, but these techniques have many limitations. One limitation of these techniques is that they require sophisticated and elaborate laboratory facilities. Another limitation of RIA is that it requires radioactive material which is health hazard and also needs trained personnel to handle the radioactive material. Still further limitation of above techniques is that reagents are expensive. Further limitation of these techniques is that minimum 4-5 hours are required to perform the tests. There is a large number of latex particle based immunoassay tests used for diagnostic purposes. The presence of antigen is often determined by the agglutination of the antibody coated particles. The tests are reliable up to a finite concentration of the antigen. Below a certain concentration of antigen, the agglutination does not occur. The agglutination is a diffusion governed process and hence quite slow. In order to enhance the rate of agglutination and improve the sensitivity of tests, especially at low concentrations in pico and femto molar range, many techniques have been tried, some of which are discussed below.
The non-cavitating standing wave ultrasound has been used to increase the sensitivity of different latex agglutination tests because of the increased rate of particle collision as antibody coated particles are forced into the pressure nodal regions (Grundy et al, J of Immunological Methods, vol 165, p 47, (1993) and Ellis et al, J Med. Microbiol., vol 49, p 853, (2000)). Microfluidic systems with controlled flow of small volumes of fluids are another approach to enhance the interaction between antigen and antibodies (Verpoorte, Electrophoresis, vol. 23, p 677, (2002) and Kricka, Clinical Chemistry, vol 44, p 2008, (1998).
Coplanar electric field has also been used to form chains of colloidal particles thereby enhancing the rate of latex agglutination reactions (Song et al, Analytical Chemistry, vol. 66, p. 778, (1994).
All the aforesaid methods of the prior art have their own inherent limitations. For example, in coplanar fields, the particles form chains. In a chain the number of neighbours is at the maximum two. In other words, it is only possible to form arrays of the particles. Hence, in case even if there are more number of binding sites on the antibody coated particle, one can not improve the specificity and sensitivity any further. Microfluidic systems need a lot of precise engineering which is not always available at hand. Thus, there is a need in the art to provide a method and system whereby the sensitivity of the assay is increased and the material in a sample may be detected even at low concentrations.
It is expressly not admitted, however, that any of the documents incorporated by reference herein teach or disclose the present invention.